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Jum/DIAGNOSTIC NUCLEAR MEDICINE jum/DIAGNOSTIC NUCLEAR MEDICINE ENDOTOXIN AS A CAUSE OF ASEPTIC MENINGITIS AFTER RADIONUCLIDE CISTERNOGRApHy JamesF. Cooper and John C. Harbert The University of Tennessee Center for the Health Sciences, Memphis, Tennessee, and Georgetown University Hospital, Washington, D.C. The role of pyrogens in aseptic meningitis after radionuclide cisternography was studied by TABLE 1. ADVERSE REACTIONS FOLLOWING means of the Limulus test, a sensitive detector RADIONUCLIDECISTERNOGRApHy of endotoxin. During a 15-month period, 39 Radiooctivetracer'Symptomatic reactions associated with cisternography were with reference meningitist reported. Ten samples of specific lots of the 1―I-IHSA Detmerand Blacker(1) radioactive drugs implicated in 20 of these re “l-IHSA Nicol (2) actions were tested and all reacted strongly “l-IHSA Oldham and Staab (3) 1311-IHSA Jonasand Braunstein(4) positive to the Limulus test. The less sensitive ‘°mTc-HSAand rabbit pyrogen test was negative for these prepa 1―l-IHSA Barnesand Fish(5) 5/50(10%) rations when tested on a dose-per-weight basis. ‘311-IHSA Messed and Rieder (6) 7/50 (14/.) ‘31l-IHSA Atkins, et al (7) 19 Our findings apparently provide clinical evi “ln-DTPA Aldersonand Siegel (8) 2/130 (1.5°!.) dence for the observation made in animals that “11n-DTPA Wagner, et al (9) 1/16(6%) endotoxin is at least 1,000 times more toxic * IHSA,radioiodinated(1311)humanserumalbumin,USP; intrathecally than intravenously. The data impli HSA, human serum albumin; DTPA, diethylenetriamine pen taaceticacid, cate endotoxin contamination as a cause of ad t Numberof patientswith symptomaticmeningitis/total verse reactions to radionuclide cisternography. number of patients in study, if reported. We conclude that the USP pyrogen test is insuffi ciently sensitive for intrathecal injectables and should be supplemented by the Limulus test. following intrathecal injection of ‘31I-IHSA (7). Adverse reactions after cisternography occurred at Georgetown Hospital in Washington, D.C., and led Radioiodinated human serum albumin (1311-IHSA) us to study their cause (11). @ and the DTPA chelates of 11n and 169Th have be Although the etiology of these adverse reactions come accepted as tracers for radionuclide cisternog was unknown, the amount of albumin (1—3,12), raphy because of their reliability in demonstrating chemical irritants (1 ,5,8) , sensitization (1 ,8) , and cerebrospinal fluid dynamics. Nevertheless, appre pyrogens (5,8,10) were advanced as possiblecauses. hension attends the use of this procedure because of The advent of the Limulus test made available a isolated reports of aseptic meningitis following intra rapid, sensitive method for detecting bacterial endo thecal ‘311-IHSAinjections (1—4). Alarmingly high toxin (pyrogens) in radiopharmaceuticals and other reaction rates, up to 14% (Table 1) , were reported parenterals (13—15). By means of the Limulus test, in recent studies, and reactions to one of the newer endotoxin was detected in radiopharmaceuticals, agents were reported (5—9). Banerji and Spencer which caused aseptic meningitis after intrathecal in (10) reported that 27% of 60 patients had a febrile jection, and this contaminant was implicated as the response of at least 1°Fwithin 24 hr of ‘311-IHSA etiologic agent in the adverse reactions reported here. cisternography; when patients had accompanying pneumoencephalograms, the incidence was 41 % of Received Jan. 30, 1975; revision accepted April 10, 1975. 88 patients. The survey of adverse reactions to radio For reprints contact: J. F. Cooper, Radiomedications Sec pharmaceuticals sponsored by the Society of Nuclear tion, Dept. of Medicinal Chemistry, The University of Ten nesseeCenter for the Health Sciences, 26 S. Dunlap, Room Medicine presented 19 reports of aseptic meningitis 107,Memphis,Tenn. 38163. Volume 16, Number 9 809 COOPER AND HARBERT MATERIALSANDMETHODS 0 and V grades are positive for bacterial endo Glassware was cleaned, sterilized, and rendered toxin. It has been our experience with endotoxin pyrogen-free by heating in a dry-air oven at 200°C solutions and small-volume parenteral products that for at least 2 hr. Sterile, pyrogen-free solutions were a positive Limulus test with a gel time greater than used for all procedures. Specimens of Limulus poly 25 mm will not produce a pyrogenic response in phemus (horseshoe crab) were collected near Chin rabbits when the same solution is injected intrave coteague, Va., and Limulus amebocyte lysate was nously at a dose of I ml/kg (13,14). On this assump prepared by a modification of the Levin method tion, positive reactions for radiopharmaceuticals are (14,15) . The amebocytes were lysed by adding Ster categorized as follows by the gel time (the time in ile Water for Injection, USP, to cells in a 2 :1 ratio minutes required for a firm gel to form after prompt by volume while agitating vigorously. The super mixing and incubation of the assay mixture) : strong nate was removed and stored at 40°C. positive, gel time less than 30 mm; positive, gel time 30—60mm; and trace, gel time greater than 60 mm Purified endotoxins of Escherichia coli (E. coli or a V grade test result. 026 :B6, Difco No. 5745 14) and Kiebsiella pneu Preparations of 1311-IHSA, radioiodmated (131!) moniae, the FDA reference pyrogen ( 16 ) , were pre human serum albumin, USP, were the products of pared in stock solutions of 100 ,@g/ml. The sensitivity Abbott Laboratories (Risa-131-H) and E. R. Squibb of lysate preparations was determined with freshly & Sons (Albumotope-1311). Preparations of 1@In prepared twofold dilutions of these endotoxins, 2 diethylenetriamine pentaacetic acid (@In-DTPA) through 0.1 25 ng/ml concentrations. Acceptable were obtained from one supplier. lysate detected 1 ng/ml of these endotoxins within 1 hr by forming a firm gel. Sodium Chloride for In jection, USP (Abbott Laboratories), or Sterile Water RESULTS for Injection, USP, was used for endotoxin stock Limulus tests were conducted on over 100 differ solutions and dilutions and served as the negative ent commercial lots of ‘311-IHSAand 1111n-DTPA control when mixed with Limulus amebocyte lysate. preparations during a 15-month period beginning in Test units of lyophilized lysate were prepared by March 1972. Test samples and notifications of ad lyophilizing 0.1 ml of tris-buffered lysate (pH 7) in verse reactions came to us from investigators and 2-ml funnel-top ampuls (Wheaton Glass Co. ) and industry or by response to a published request for sealing with rubber closures under vacuum. The participation in this study (17). Of 39 cases of lyophilized lysate was stored below 0°Cor at 4°C aseptic meningitis, we obtained 10 samples of radio and assayed as described above. A slight increase pharmaceuticals for assay implicated in 20 adverse in sensitivity was often observed for the lyophilized patient reactions. Every lot of 1311-IHSA and 1111n- units. This was attributed to the fact that the relative DTPA tested associated with an adverse reaction endotoxin concentration was higher in the test am yielded a strongly positive result by the Limulus test. puls when reconstituted with the test solution than One reaction occurred after Risa-1 3 1-H intra when mixed in equal parts with liquid lysate. A com thecal injection but the specific lot was unavailable mercial Limulus amebocyte lysate (Pyrogent, by for a Limulus test. However, 35 of the lots of this Mallinckrodt, Inc. ) was used as directed by the drug tested by the Limulus test during this period package insert in a comparative study. were either negative or contained trace levels of The Limulus test was conducted by injecting 0.25W- endotoxin. 0.3 ml of the radiopharmaceutical directly into a Of 10 lots of Albumotope-131I associated with 24 test ampul of lyophilized lysate. The assay mixture reactions, 5 lots were obtained that were implicated was thoroughly and quickly mixed and then incu in 8 adverse reactions. All five reacted strongly posi bated immediately in a water bath at 37°C. Test tive with the Limulus test (Table 2). Another lot ampuls were observed periodically without further tested during this time was also strongly positive. agitation for at least 60 mm for the formation of an One lot of Albumotope-1311 (assay date of July opaque gel. The time required for gel formation 21 , 1972, Table 2), which was both Limulus-test was recorded in minutes as the time of gelation. The positive and associated with untoward effects, was results were graded in our laboratory as follows: assayed after serial dilutions to approximate the G, formation of a firm gel that remains intact when endotoxin concentration. The undiluted solution and inverted slowly to 180°; V, increase in viscosity, 1 : 10 and 1 : 100 dilutions (in Sodium Chloride for opacity, and observance of gelatinous granules; and Injection, USP) gave 12-, 15-, and 25-mm gel times, N, negative,no formation of opaque gelatinousgran respectively, by the Limulus test. These results mdi ules. cated a high level of endotoxin contamination in the 810 JOURNAL OF NUCLEAR MEDICINE DIAGNOSTIC NUCLEAR MEDICINE 10 ,@g/ml. Therefore, inadequate column washing TABLE 2. ADVERSE REACTIONS TO could have contributed significant amounts of endo RADIONUCLIDE CISTERNOGRAPHYAND toxin to these radioiodinated drugs. LIMULUS TEST RESULTSFOR ALBUMOTOPE.131l* We learned of 14 reactions to the ‘‘‘In-DTPA AdversereactionsAssay preparations. Five were isolated events, but nine dote for lot CasesHospitalsLimulus testst reactions followed the use of two specific lots in May 1973. The manufacturer supplied samples of these April 28, 1972 2 2 NA June 2, 1972 2 2 NA two lots and all solutions associated with their formu June 9, 1972 2 G-12 lation as well as three other implicated lots. All five July 14, 1972 G-12 lots of “‘In-DTPAwere found positive by the Limu July 21, 1972 2 2 G-1 2 July 28, 1972 7 2 NA lus test.
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